Diagnostic assay for breast cancer susceptibility

ABSTRACT

The lifetime probability of a woman developing breast cancer can now be determined based on an allelic variation found in the 3′UTR of the prohibitin gene. The probability is dependent on the sequence of the 3′UTR at position 729, i.e., whether there is a thymine (T) or a cytosine (C) or both at this position. Polymorphism at position 729 is also disclosed as a susceptibility indicator for hereditary breast cancer in men. Determining the sequence at the position 729 can be done by any number of standard techniques. Preferably, the sequence is determined by amplifying this region by PCR and subjecting it to an RFLP analysis.

This Application is a 371 of PCT/US97/20844, filed on Nov. 6, 1997 andclaims benefit of 60/029,978 filed on Nov. 7, 1996.

TECHNICAL FIELD OF THE INVENTION

The invention relates to a diagnostic assay for determiningsusceptibility to breast cancer based on the sequence of the 3′untranslated region of the prohibitin gene.

BACKGROUND OF THE INVENTION

Breast cancer is the second leading cause of cancer-related deaths ofwomen in North America. A distinction must be drawn, however, betweensporadic and familial or inherited breast cancer. Approximately 10% ofall breast cancers are currently classified as strongly familial withmany of these appearing to be caused by mutations in the hereditarybreast cancer genes BRCA1 or BRCA2. However, at least one-third ofbreast cancers which seem to run in families are not linked to BRCA1 orBRCA2, suggesting the existence of an additional hereditary breastcancer gene or genes. Recently, studies have suggested the possibilitythat additional genes important for breast cancer development arelocated on chromosome 17, based on the observation of tumor suppressionin breast cancer cells following the introduction of normal humanchromosome 17 without the inclusion of active BRCA1 or p53 (Theile, etal., “Suppression of tumorigenicity of breast cancer cells by transferof human chromosome 17 does not require transferred BRCA1 and p53genes,” Oncogene 10:439-443 (1995)).

The antiproliferative gene prohibitin was discovered using a subtractionhybridization to enrich for mRNAs preferentially expressed in normallyproliferating cells compared to regenerating rat liver cells (McClung,et al., “Isolation of a cDNA that hybrid selects antiproliferative mRNAfrom rat liver,” Biochem Biophys Res Comm 164:1316-1322 (1989); Nuell,et al., “Prohibitin, an evolutionarily conserved intracellular proteinthat blocks DNA synthesis in normal fibroblasts and HeLa cells,” MolCell Biol 11:1372-1381 (1991)). The human prohibitin gene, which maps tochromosome 17 at q21 (White, et al., “Assignment of the human prohibitingene (PHB) to chromosome 17 and identification of a DNA polymorphism,”Genomics 11:228-230 (1991)), was an initial candidate gene for thefamilial breast and ovarian tumor suppressor locus based on a frequentloss of heterozygosity in this region in familial and sporadic breastcancers (Black, et al., “A somatic cell hybrid map of the long arm ofhuman chromosome 17, containing the familial breast cancer locus(BRCA1),” Am J Hum Genet 52:702-710 (1993); Nagai, et al., “Detaileddeletion mapping of chromosome segment 17q12-21 in sporadic breasttumors,” Genes, Chromosome and Cancer 11:58-62 (1994)). Furthermore,Sato, et al., “The human prohibitin gene located on chromosome 17q21 ismutated in sporadic breast cancer,” Cancer Res 52:1643-1646 (1992),reported four mutations in a highly conserved region of prohibitin exon4 in an analysis of 23 sporadic human breast cancers. However,positional cloning studies resulted in the identification of BRCA1rather than prohibitin (Miki, et al., “A strong candidate for the breastand ovarian cancer susceptibility gene BRCA1,” Science 266:66-71 (1994))as a familial breast cancer gene on chromosome 17.

Previous studies of the prohibitin gene from DNA purified from familialbreast cancer patients provided no evidence that any of the patientscarried a germline change of the protein coding region of prohibitingenomic DNA. It was therefore concluded that there was no relationshipbetween familial/hereditary breast cancer and mutations in theprohibitin protein coding region. Tokino, et al., Internat'l J Oncol3:769-772 (1993). Additional studies did not identify any somaticmutations in the prohibitin protein coding region in familial/hereditarybreast cancers suggesting that the protein coding region is notfrequently mutated in breast cancers. Sato et al., Genomics 17:762-764(1993).

Previous work by Jupe et al. disclosed a diagnostic test for increasedsusceptibility to sporadic breast cancer. It was reported thatindividuals who are heterozygous for the two prohibitin alleles(designated as “non-B” and “B” based on sequence variations found inintron 2 and 5) or homozygous for non-B allele would have low risk fordeveloping sporadic cancer. The probability of developing cancer wouldincrease for those who are homozygous for the B-type allele and againfor those who have a mutation in the 3′ untranslated region (“3′UTR”) ofat least one of the B-type alleles. Analyses were reported ofbreast-cancer derived cell lines and primary breast tumors showinghomozygosity for the B-type allele and somatic mutations in the 3′UTR.

Full length prohibitin cDNAs for the BT-20, MCF7 and SK-BR-3 breastcancer cell lines were sequenced, and mutations restricted to the 3′UTRwere identified. These three cell lines were arrested in cell cycleprogression when full length prohibitin transcript was introduced bymicroinjection. All of them were also homozygous for the B-allele.Compared to the sequence of the wild type prohibitin 3′UTR, two pointmutations were identified for BT-20: G (guanine) to A (adenine) atposition 758 and T (thymine) to C (cytosine) at position 814. MCF7 alsohad two point mutations: G to A at position 236 and C to T at position729. SK-BR-3 showed 26 base changes including a change of C to T atposition 729. Thus, MCF7 and SK-BR-3 both had a change of C to T atposition 729. Jupe, et al., “Prohibitin in breast cancer cell lines:Loss of antiproliferative activity is linked to 3′ untranslated regionmutations,” Cell Growth and Differentiation 7:871-878 (1996).

It has now been found, contrary to the teachings of the prior prohibitinwork, that this change from C to T at position 729 is the result not ofa somatic mutation, but rather the result of a natural allelic variationat this point, i.e., it is a germline polymorphism. Furthermore, it is agermline polymorphism that can be used as a susceptibility marker forbreast cancer. Data indicate that the frequency of homozygosity for729-T appears to be approximately 4-5-fold higher in breast cancerpatients than in unaffected females, that 4% of all breast cancersdevelop in women who are homozygous T/T (which likely make up less than1% of unaffected women), and that their lifetime risk of developingbreast cancer is approximately 50%.

Thus, it has now been found that the prohibitin gene, located onchromosome 17q21 near the BRCA1 locus, exhibits a germline polymorphismin the 3′UTR that can be used as a susceptibility marker for breastcancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A and FIG. 1B show the alignment of mutated prohibitin 3′UTRsequences obtained from micro-dissected patient tumors. The figures showthe sense DNA strand of a frequently mutated 140 bp stretch located atthe 3′ end of the 3′UTR, with the first 70 bases given in FIG. 1A andthe last 70 bases given in FIG. 1B. The sequences of the two wild-type(WT) alleles which differ only at position 729 are shown at the top. Themutated sequences from seven different patient (TN-X) breast tumors (T)are aligned with the sequences obtained from adjacent normal (N) tissue.These adjacent normal sequences were all identical except at position729. This position contained either a C or T (C/T). The sequence fromthe heavily mutated breast cancer cell line, SK-BR-3 is shown in thecenter of the figures. The small case letters depict base changesrelative to the wild type sequence. Insertions are shown by additionalsmall case letters and deletions are depicted by dashes. The variantbase at UTR-729 is enclosed in the shaded box and the six base AflIIIrecognition site is enclosed in the large box.

FIG. 2 illustrates the 5′−3′ sense sequence of the wild type prohibitin3′UTR and the location of primers (underlined) which may be utilized foran AflIII restriction fragment length polymorphism (RFLP) assay forgenotyping. The assay is run in two steps with the initial primer setP1/P2 being used for PCR amplification. The initial PCR reactionproducts are then run on a 2.5% agarose gel and the 852 bp band isexcised and purified. The 852 bp fragment is used as the template in PCRwith one of the primer sets P3/P2 or P4/P2 to produce a sub-fragment.This subfragment is purified through microspin columns (Pharmacia), anddigested with AflIII. Primers P1, P3, and P4 are all sense primers.Primer P2 is an antisense primer whose sequence is5′-GGAAGGTCTGGGTGTCATTT-3′ (SEQ ID NO:19).

FIG. 3 illustrates the 5′−3′ sense sequence of the prohibitin gene whichbegins in intron 6, contains the protein coding region of exon 7 andcontinues to the end of the 3′UTR. The primers P1′ (SEQ ID NO:23)(forward) and P2 (SEQ ID NO:19) (reverse) are used to synthesize the PCRfragment that is used for an AflIII RFLP genotyping assay. The 1237 bpfragment (from position 93 to position 1328 in FIG. 3) that issynthesized is digested with AflIII to determine the genotype. Thesymbol “*” marks the beginning of the 852 bp 3′UTR coding sequence (FIG.2), and the numbers on the right of the sequence give the base numberfor the 852 bp 3′UTR coding sequence. The symbol “++++++” shows thelocation of the constitutive AflIII site while the symbol “******” showsthe location of the polymorphic site. Cleavage by AflIII is lost whenthe site is ATGTGT. The C to T polymorphism occurs at position 729 inthe 852 bp UTR (FIG. 2) and position 1205 in this sequence (FIG. 3).Also shown on this figure are the forward P3′ primer (SEQ ID NO:25) andreverse P4′ primer (SEQ ID NO:26) used to synthesize the 442 bp probeused in Southern blotting experiments as described in Example 1.

FIG. 4 illustrates the diagnostic restriction fragment lengthpolymorphism analysis (RFLP) patterns obtained with the PCR assay asdescribed in FIG. 3. The genotypes illustrated are as follows: 1-C/C;2-C/T; 3-T/T. The sizes of the fragments observed are shown to the leftof the figure. The 671 bp fragment is common to all genotypes. Thepattern shown for the 566 bp and 442 bp fragments is also observed withgenomic Southern blots using the 442 bp probe.

DETAILED DESCRIPTION OF THE INVENTION

Based on the frequencies of the C/C, C/T, and T/T germline genotypes atposition 729 (as defined in FIG. 2 of the application) in the prohibitin3′UTR among controls and breast cancer cases, a simple test has beendeveloped to determine the susceptibility of lifetime probability of anindividual developing breast cancer. Given that the overall probabilityof a woman developing breast cancer in the U.S. is 12.5%, Bayes Theoremwas applied to the genotypic frequencies determined on 72 breast cancerpatients and 92 unaffected females (Table 1), and the resultingprobability that a woman with a particular genotype will develop breastcancer over the course of her lifetime {probability=[(frequency ofbreast cancer patient having given genotype)×(overall probability of awoman developing breast cancer, i.e., 0.125)]±[frequency of unaffectedwoman having given genotype]×100} is as follows: C/C, 10%; C/T, 17%; andT/T, approximately 50%. While the calculated percentages may varydepending on the size of the sampled population, it is expected that thedisclosed percentages will provide a useful guide as to risk.

To determine a woman's probability of developing breast cancer requiresonly a determination of the woman's germline prohibitin genotype withregard to position 729 of the 3′UTR; that is, whether the woman ishomozygous thymine (T/T) or homozygous cytosine (C/C) or heterozygous(C/T) at position 729. To determine an individual's genotype at position729, genomic DNA can be isolated from a wide variety of patient samplesusing standard techniques. Preferably, the genomic DNA is isolated fromeither blood or buccal cell smears as described in Example 1. Followingpreparation of genomic DNA, the region containing base 729 of theprohibitin 3′UTR may be amplified, or the genomic DNA may be directlydigested (Example 1). Like the preparation of genomic DNA, this too canbe done by a wide variety of standard techniques. Preferably, thisregion is amplified by polymerase chain reaction (“PCR”) techniques, asdescribed in Example 1.

Preferably, following PCR amplification, a restriction fragment lengthpolymorphism (“RFLP”) analysis is conducted as described in Example 1.This analysis is based on the fact that the substitution of a T for C atposition 729 in the 3′UTR results in the loss of cleavability by therestriction endonuclease AflIII at its six base recognition site whichspans position 729.

Alternatively, the PCR amplified sequence at position 729 could bedetermined by any other means for distinguishing sequence variants suchas by direct sequencing using AmpliCycle™ PCR kit(Perkin Elmer) orSouthern blotting.

The probability that a particular genotype will result in breast canceris as previously stated. For those who are homozygous for the C-allele(C/C) at position 729, the probability of developing breast cancer is10%, for those who are heterozygous (C/T) the probability is 17%, andfor those who are homozygous for the T-allele (T/T) the probability of awoman developing breast cancer during her lifetime is approximately 50%.These probabilities are based on the germline prohibitin genotypes of 92control females and 72 breast cancer cases as shown in Table 1.

Being able to accurately determine a woman's genotype with respect toposition 729 serves a variety of useful purposes. First and foremost, asalready described above, it provides a means by which a woman's lifetimeprobability of developing breast cancer can be determined. For those whoare diagnosed as having an increased risk, an enhanced awareness of theincreased risk in conjunction with more frequent examinations may leadto an earlier detection of the cancer and an increased chance ofsurvival. This would be particularly useful for the newborn to those upto the age of 40 who are generally not yet screened for the developmentof breast cancer.

The assay could also be used in genetic counseling. Where the parentsare both homozygous for the T-allele, the probability of having a childwith the T/T genotype is 100%. Conversely, where the parents are bothhomozygous for the C-allele, the probability of having a child with theT/T genotype is 0%. Where only one parent is homozygous for the Tallele, or where one or both parents are heterozygous (C/T) at position729, the probability of having a child with the T/T genotype issomewhere between these two extremes and can be determined according toclassic Mendelian genetics. Depending on their genotypes, the parents ofa child could then determine the child's genotype as a newborn or evenprenatally. This information could then be used as described above todetermine an optimum schedule of examinations to ensure early detectionand treatment.

This assay could also be used for breast cancer prognosis, theprediction of disease-free interval, long-term survivorship, anddetermination of therapy for both women and men.

Determining the Prohibitin Polymorphism at UTR-729

A thorough mutation analysis was first conducted on seven individualpatients in which all of the tumors analyzed were diagnosed byhistopathology as invasive ductal carcinomas. In order to avoid crosscontamination, these analyses were done on tumor tissue and normaladjacent tissue isolated separately by microdissection from hematoxylinand eosin (H&E) stained sections. Our unpublished studies of theprohibitin 3′UTR in breast cancer cell lines and other cancer cell linesshowed that the final 200 bases contained prohibitin's antiproliferativeactivity and was also the most heavily mutated. Thus, only the final 140bp of the 3′UTR was examined. PCR amplication products were cloned intothe TA cloning vector, and at least two clones from the tumor and twoclones from normal adjacent tissue were sequenced from each patient.FIGS. 1A and 1B show an alignment of these seven tumor sequences[TN-56(T), SEQ ID NO:2; TN-78(T), SEQ ID NO:4; TN-50(T), SEQ ID NO:6;TN-1(T), SEQ ID NO:9; TN-3(T), SEQ ID NO:11; TN-31(T), SEQ ID NO:13; andTN-94(T), SEQ ID NO:15] and adjacent normal tissue [TN-56(N), SEQ IDNO:3; TN-78(N), SEQ ID NO:5; TN-50(N), SEQ ID NO:7; TN-1(N), SEQ IDNO:10; TN-3(N), SEQ ID NO:12; TN-31(N), SEQ ID NO:14; and TN-94(N), SEQID NO:16)] compared to the wild-type prohibitin 3′UTR (WT) (SEQ ID NO:1)and the heavily mutated breast cancer cell line SK-BR-3 (SEQ ID NO:8)sequences in a 140 b.p. region near the 3′ end of the wild-typeprohibitin 3′UTR (SEQ ID NO:17).

The most frequently and only consistently altered site was the cytosine(C) at position 729 which was changed to a thymine (T) in all of thetumors. The 3′UTR sequences from the adjacent normal tissue from each ofthe patients is also shown directly beneath the tumor sequences in FIGS.1A and 1B. This analysis showed that all of the normal tissue waswild-type except for demonstrating heterozygosity at position 729 of the3′UTR.

The finding that seven out of seven normal tissue specimens adjacent tobreast tumors showed heterozygosity (C/T) at position 729 in theprohibitin 3′UTR was unexpected, as prior to this time, only “C” hadbeen found at this position in normal tissue or cell lines derived fromnormal tissue. Further studies were then performed to determine if the Twas an “initiator” mutation responsible, at least in part, for thedevelopment of the tumor, but also found in histologically normaladjacent tissue indicating a clonal or field effect, or if the Trepresented a germline allelic variant (polymorphism) present in thenormal population. To distinguish between the two possibilities, testswere done to determine if there were thymines at position 729 ingermline tissue far removed from the tumor in breast cancer patients andin control individuals. That is, the frequency of T vs C at position 729of the 3′UTR (UTR-729) was determined in tissue representing thegermline genotype (blood sample and/or buccal cell scrapes) from thesetwo groups.

The region containing UTR-729 is cleaved by the restriction enzymeAflIII when cytosine (C) is present in the wild type sequence. Thiscleavage site is lost when a thymine (T) is present. Sequencing frompatient samples identified only this single change within the AflIIIrecognition site (FIGS. 1A and 1B). In an AflIII RFLP analysis, 70% of92 healthy female volunteers were

TABLE 1 Genotype and allele frequencies of 3'UTR variants among controlsand breast cancer cases Number Frequency Breast Cancer Control FemalesPatients Genotypes C/C 64 0.696 41 0.569 C/T 27 0.293 28 0.389 T/T  10.011  3 0.042 Total 92 1.000 72 1.000 Alleles C 155  0.842 110  0.764 T29 0.158 34 0.236 Total 184  1.000 144  1.000

homozygous C/C, 29% were heterozygous C/T, and 1.1% were homozygous T/T(Table 1). This is in contrast to 72 breast cancer patients where 4%were T/T, 39% were C/T and 57% were C/C (χ²=3.672 and p<0.159). Assumingthat the data in Table 1 accurately reflect the genotypic frequencies ofcases and controls in the general population and that the lifetime riskof breast cancer is 12.5%, Bayes' Theorem (Fleiss, J., StatisticalMethods for Rates and Proportions, 2nd ed., John Wiley & Sons, New York,1981, pp. 1-17) can be used to calculate the probability that a womanwith a particular genotype will develop breast cancer over the course ofher lifetime. These probabilities are: 10% for C/C, 17% for C/T, andapproximately 50% for T/T. Furthermore, if 4% of all breast cancer casesare T/T, then 4% of women who will eventually get breast cancer can beidentified by this simple test.

The mean age ± standard deviation for the control and cases were 40±12.9and 55±11.5 years, respectively. The majority of the cases and controls(95%) are Caucasian females residing in Oklahoma. Potential relativerisk of breast cancer was also examined in terms of the odds ratio (OR).The odds ratio for subjects having C/T and subjects having T/T combined,i.e., T carrier, was calculated as OR_(T)=[(number of breast cancerpatients having C/T+number of breast cancer patients having T/T)±(numberof breast cancer patients having C/C)]±[(number of unaffected subjectshaving C/T+number of unaffected subjects having T/T)±(number ofunaffected subjects having C/C)]. The odds ratio for subjects havingT/T, i.e., homozygous T, was calculated as OR_(T/T)=[(number of breastcancer patients having T/T)±(number of breast cancer patients havingC/C+the number of breast cancer patients having C/T)]±[(number ofunaffected subjects having T/T)±(number of unaffected subjects havingC/C+the number of unaffected subjects having C/T)]. The odds ratiocalculated after combining subjects carrying either C/T or T/T is about1.7. When T/T is considered separately with C/T and C/C combined, theodds ratio is about 4.0. Again, while the calculated odds ratios mayvary depending on the size of the sampled population, it is expectedthat the disclosed ratios will provide a useful guide as to risk.

Relevance of Polymorphism at UTR-729 to Cancer

In the U.S., a woman has a 1 in 8 (12.5%) risk of developing breastcancer during her lifetime and a 1 in 28 (3.6%) risk of dying from thedisease (Boring, et al., “Cancer statistics,” CA Cancer J Clin 44:7-26(1994)). Approximately 10% of all breast cancers are currentlyclassified as familial and many of these appear to be caused by germlinemutations in the BRCA1 gene on chromosome 17q21 (Hall, et al., “Linkageof early onset familial breast cancer to chromosome 17q21,” Science250:1684-1689 (1990)) or the BRCA2 gene on chromosome 13q12-13 (Wooster,et al., “Localization of a breast cancer susceptibility gene, BRCA2, tochromosome 13q12-13,” Science 265:2088-2090 (1994)). However, the vastmajority of breast cancers are considered to be sporadic. It wasoriginally thought that many of these sporadic cancers would also becaused by somatic mutations in familial breast cancer genes. This hasnot proven to be the case. Few, if any, somatic mutations of BRCA1 havebeen found in sporadic breast tumors (Merajver, et al, “Somaticmutations in the BRCA1 gene in sporadic ovarian tumours,” Nat Genet9:439-443 (1995); Hosking, et al., “A somatic BRCA1 mutation in anovarian tumour,” Nat Genet 9:343-344 (1995); and Futreal, et al., “BRCA1mutations in primary breast and ovarian carcinomas,” Science 266:120-122(1994)). In other studies, only one out of 70 (Lancaster, et al., “BRCA2mutations in primary breast and ovarian cancers,” Nat Genet 13:238-240(1996)), and one out of 100 breast cancer patients had somatic mutationsin BRCA2 (Miki, et al., “Mutation analysis in the BRCA2 gene in primarybreast cancers,” Nat Genet 13:245-247 (1996); Teng, et al., “Lowincidence of BRCA2 mutations in breast carcinoma and other cancers,” NatGenet 13:241-244 (1996)). Thus, mutations at these loci do not appear tobe important in the majority of sporadic breast cancers.

Table 1 suggests that women who carry even a single germline prohibitinallele with the 729-T polymorphism are at approximately 2.0-foldincreased risk for breast cancer compared to those who are homozygousfor 729-C (17% vs. 10%). Furthermore, Bayes' Theorem predicts that womenwho are homozygous at 729-T have a 50% risk of developing breast cancerover the course of a lifetime. If the data in Table 1 are representativeof all breast cancers, then approximately 4% of breast cancer cases willdevelop in T/T women even though they represent less than 1% of thetotal population. Therefore, screening women in the general populationfor this polymorphism will allow the identification of up to 1% of allwomen with a significantly higher than average risk of eventuallygetting breast cancer. Likewise, women who are homozygous C/C (70% ofthe total population) can be counseled that their probability ofdeveloping breast cancer over their lifetime is approximately 10%, oronly slightly below the average risk. Furthermore, the 729-Tpolymorphism is inherited, which suggests that a substantial subset ofbreast cancers previously considered to be sporadic (i.e., thosedeveloping in C/T and T/T women or 43% overall), have a hereditarycomponent. From the relative proportions of C/C, C/T, and T/Tindividuals in the general population (Table 1), it can be assumed thatmost homozygous T/T women have heterozygous C/T parents. In this case,the probability of a T/T woman with breast cancer having a sibling whois also T/T is 1 in 4. This is precisely the risk of developing breastcancer for women who have first degree relatives with the disease(Boring, et al., CA Cancer J Clin 44:7-26 (1994)).

Example 1 Diagnostic Assay Methodology

The diagnostic assay for determining susceptibility of breast cancerbased on the sequence of the 3′UTR of the prohibitin gene is describedbelow.

Sample Collection

Blood samples (approx. 10 ml) were collected by routine venipunctureinto tubes containing anticoagulant.

Buccal cell smears were collected using sterile cytology brushes (typeH—Histobrush, 174-600; Spectrum Laboratories, Dallas, Tex.). The studyparticipant was instructed to twirl the brush on the inner cheek for 30seconds on each side. The brush was then inserted into a sterilecollection tube, tightly capped, and stored at 4° C. prior to DNAtemplate preparation.

DNA Preparation

The DNA from blood samples was prepared using the PureGene Kit (Gentra,Minneapolis, Minn.).

The DNA from buccal cell smears was isolated using a method described byHorrigan, et al., “Polymerase chain reaction-based diagnosis of Del(5q)in acute myeloid leukemia and myelodysplastic syndrome identifies aminimal deletion interval,” Blood 88:2665-2670 (1996), which is amodification of a method originally published by Richards, et al.,“Multiplex PCR amplification from the CFTR gene using DNA prepared frombuccal brushes/swabs,” Hum Mol Genet 2:159-160 (1993). The cytologybrush was transferred to a 1.5 ml tube containing 0.6 ml of 50 mMsterile NaOH. The handle of the brush was clipped, and the lid wasclosed. After vortexing for 30 seconds, the sample was heated to 95° C.for 5 minutes. The tube was vortexed again, and the brush was drained torecover residual liquid prior to removal from the tube. The solution wasneutralized by adding 0.06 ml of 1 mM Tris, pH=8.0. After thoroughmixing, the sample was stored at −20° C. The assay can also be performedon high molecular weight DNA purified from skin, hair follicles, andvirtually any other tissue source as well as from fibroblast orlymphoblast cell lines. In this case, the DNA can be prepared using thePureGene kit (Gentra, Minneapolis, Minn.), or any similar method, inaccordance with the manufacturer's instructions.

Polymerase Chain Reaction

PCR reactions were run on 0.1 μg of genomic DNA purified from blood or0.010 ml of buccal smear extract using Taq Gold polymerase (PerkinElmer, Foster City, Calif.). The reaction conditions used were asfollows: 10 mM Tris-HCl, pH=8.0, 50 mM KCl, 1.5 mM MgCl, 100-200 μM eachof DATP, dGTP, dTTP, and dCTP, 0.1% Triton X-100, 0.5-1.0 units Taq Goldpolymerase, and 100 ng of each primer in a 50-μl reaction mix.

In one form of the assay, as illustrated in FIG. 2 and SEQ ID NO:17, an852 bp 3′UTR synthesized with primers 5′-CCCAGAAATCACTGTG-3′ (primer P1,sense) (SEQ ID NO:20) and primer P2 (SEQ ID NO:19) is gel purified and asecondary PCR product is synthesized using the primers5′-TGAGTCCTGTTGAAGACTTCC-3′ (primer P3, sense)(SEQ ID NO:18) and5′-GGAAGGTCTGGGTGTCATTT-3′ (primer P2, antisense)(SEQ ID NO:19).

Restriction Fragment Length Polymorphism Analyses

The PCR products were digested with the restriction enzyme AflIII usingthe buffer and conditions recommended by the manufacturer (New EnglandBiolabs, Cambridge, Mass.). All digestions for a group of individualsamples were performed using a diluted master mix. Controls withconfirmed sequence were included with each series of digests. Thedigestion products were separated by electrophoresis on 20% acrylamidegels, stained with ethidium bromide and visualized by ultraviolet light.

Alternatively, high molecular weight DNAs purified by using the PureGenekit were analyzed for restriction fragment length polymorphisms bySouthern blotting. Generally, 10-15 μg of DNA was digested with therestriction enzyme AflIII (New England Biolabs) at 37° C. for 16 hoursusing the manufacturer supplied buffer. The digests were terminated byprecipitating the DNA by adding {fraction (1/10)} volume 3M sodiumacetate and 2 volumes of absolute ethanol. Following resuspension inwater and addition of loading dye (Promega 6X), the samples were loadedinto a 1% agarose gel, and electrophoresis was performed until thebromophenol blue loading dye reached the bottom of the gel. Gels werethen denatured in 0.5 M NaOH/1.5M NaCl for 30 minutes followed byneutralization in 0.5M Tris/1.5 M NaCl (pH=7.0). A Southern blot wasthen carried out by capillary transfer to Hybond membrane (Amersham,Arlington Heights, Ill.). The DNA was fixed to the membrane either bybaking at 80° C. or crosslinking with ultraviolet light.

The RFLP was detected by probing with a nucleic acid fragment containingthe prohibitin 3′UTR. The routinely used probe was a 442 bp nucleic acidfragment that lies immediately 5′ to the polymorphic AflIII cut site. Itwas synthesized by PCR using a full length 3′UTR clone for template andprimers P3′ and P4′ (FIG. 3). The probe was labeled using a randomprimer labeling kit (Pharmacia, Piscataway, N.J.). The membranes werehybridized at least 12 hours at 65° C. and washed at the sametemperature under high stringency. The filter was then exposed to x-rayfilm or a phosphoimager screen to display the RFLP for interpretation.Alternatively, a 124 bp fragment 3′ to the polymorphic AflIII site, aswell as the 566 bp fragment synthesized with P3′ and P2 primers (FIG. 3)may be used as a probe. Any of these probes will display an RFLP thatdistinguishes the different genotypes. Southern blots probed with the442 bp probe displayed the 566 bp and 442 bp banding pattern shown inFIG. 4.

The substitution of a T for C at position 729 (FIG. 2) in the 3′UTRresults in the loss of cleavability by AflIII at its six baserecognition sequence. Our analyses of mutated breast tumors (7), breastcancer cell lines (3), and buccal cell scrapes from homozygous T breastcancer patients (7) show that the C to T at 729 is the only change inthe recognition site thus far detected that is responsible for loss ofAflIII cutting. Homozygous C individuals have both alleles cut at thepolymorphic site, while alleles of homozygous T individuals do not cut.Heterozygous individuals have one allele of each, C and T.

Example 2 Alternative Diagnostic Assay Method

An alternative assay was performed as given in Example 1, with theexception that the secondary PCR product was synthesized using the senseprimer P4, 5′-GGATGGACTTGTATAG-3′ (SEQ ID NO:21) and the antisenseprimer 5′-GGAAGGTCTGGGTGTCATTT-3′ (primer P2, antisense)(SEQ ID NO:19).

Example 3 Alternative Diagnostic Assay Method

An alternative assay was performed as given in Example 1, with theexception that, as illustrated in the 1237 bp genomic sequence given inFIG. 3 and SEQ ID NO:22, the primers utilized were5′-AAGGTGGCTTTCTGGTGAAG-3′ (primer P1′, sense)(SEQ ID NO:23) and5′-GGAAGGTCTGGGTGTCATTT-3′ (primer P2, antisense)(SEQ ID NO:19). In thisassay using SEQ ID NO:22, the base at position 1205 corresponds to theposition 729 in SEQ ID NO:17.

FIG. 4 illustrates the pattern of bands produced in this assay for eachgenotype. Utilizing the sense primer SEQ ID NO:23 and antisense primerSEQ ID NO:19, the RFLP pattern for a homozygous C individual (C/C) showsthat for both DNA strands, the 566 bp measured from the constitutiveAflIII site to the end of the 3′UTR was cut at position 729/1205 intotwo distinct bands of 442 bp and 124 bp. A homozygous T individual (T/T)produced one band of 566 bp measured from the constitutive AflIII siteto the end of the 3′UTR which was uncut at position 729/1205 on both DNAstrands. The heterozygous individual (C/T) gave three distinct bands,showing that for one DNA strand, the 566 bp measured from theconstitutive AflIII site to the end of the 3′UTR was cut at position729/1205 into two distinct bands of 442 bp and 124 bp, and for the otherDNA strand, one band of 566 bp measured from the constitutive AflIIIsite to the end of the 3′UTR was uncut at position 729/1205. In thisassay, a band common to all genotypes is the 671 bp fragment measuredfrom the 5′ end of the PCR product to the constitutive cut site.

This method is a single step process that shows 100% correlation withSouthern blot results.

Example 4 Alternative Approaches

The predictive value of this assay involves determining the germlinegenotype of an individual at position 729 in the prohibitin 3′UTR. Thereare many potential specific methods that can be used to accomplish thistask. We have primarily used the RFLP described in Example 1 and DNAsequencing to collect our data. However, any other methods based onsingle base oligonucleotide mismatch screening (Jupe, E. R. and Zimmer,E. A., “Assaying differential ribosomal RNA gene expression withallele-specific oligonucleotide probes,” In Methods inEnzymology-Molecular Evolution: Producing the Biochemical Data, AcademicPress, pp. 541-552, 1993), allele specific PCR amplification (Allen, etal., BioTechniques 19:454 (1995); Ault, G., J Virological Methods46:145-156 (1994); Tada, M., Cancer Research 53:2472-2474 (1993); Huang,Nucleic Acids Research 20:4567-4573 (1992); Sommer, BioTechniques12:82-87 (1992); and Kwok, Nucleic Acids Research 18:999-1005 (1990)),or a method employing a high specificity thermostable ligase (Ampligase,Epicenter Technologies) could be applied for detection of thepolymorphism. In addition, any method currently in use such as singlestrand conformation polymorphisms or denaturing gradient gelelectrophoresis, or any method developed in the future for detectingsingle base changes, could also be applied to the detection of thesegenotypes. This test could also be performed starting with RNA. In thiscase, the RNA would be analyzed directly by sequencing or converted tocDNA using reverse transcriptase (Castles, et al., BioTechniques21:425-428 (1996), followed by PCR and any method capable of detectingsingle base changes.

Example 5 Diagnostic Assay for Hereditary Breast Cancer in Men

The C/T polymorphism at position 729 in the prohibitin 3′UTR is alsouseful for the diagnosis of susceptibility to breast cancer in men.

A portion of genomic DNA isolated from a male patient diagnosed withbreast cancer was examined according to the assay given in Example 3.The patient's genotype was identified as T/T, which corresponds to anincreased risk for breast cancer.

25 140 base pairs nucleic acid double linear DNA (genomic) Homo sapiensmisc_feature 1..140 /note= “wild type (WT)” 1 AGTGGAATTC CAACTTGAAGGATTGCATCC TGCTGGGGCT GAACATGCCT GCCAAAGAYG 60 TGTCCGACCT ACGTTCCTGGCCCCCTCGTT CAGAGACTGC CCTTCTCACG GGCTCTATGC 120 CTGCACTGGG AAGGAAACAA140 140 base pairs nucleic acid double linear DNA (genomic) Homo sapiensmisc_feature 1..140 /note= “TN-56 (tumor)” 2 AGTGGAATTC CAACTTGAAGGATTGCATCC TGCAGGGGCT GAACATGCCT GCCAAAGATG 60 TGTCCGACCT ACGTTCCTGGCCCCCTCGTT CAGAGACTGC CCTTCTCACG GGCTCTATGC 120 CTGCACTGGG AAGGAAACAA140 140 base pairs nucleic acid double linear DNA (genomic) Homo sapiensmisc_feature 1..140 /note= “TN-56 (normal)” 3 AGTGGAATTC CAACTTGAAGGATTGCATCC TGCTGGGGCT GAACATGCCT GCCAAAGAYG 60 TGTCCGACCT ACGTTCCTGGCCCCCTCGTT CAGAGACTGC CCTTCTCACG GGCTCTATGC 120 CTGCACTGGG AAGGAAACAA140 140 base pairs nucleic acid double linear DNA (genomic) Homo sapiensmisc_feature 1..140 /note= “TN-78 (tumor)” 4 AGTGGAATTC CAACTTGAAGGATTGCATCC TGCAGGGGCT GAACATGCCT GCCAAAGATG 60 TGTCCGACCT ACGTTCCTGGCCCCCTCGTT CAGAGACTGC CCTTCTCACG GGCTCTATGC 120 CTGCACTGGG AAGGAAACAA140 140 base pairs nucleic acid double linear DNA (genomic) Homo sapiensmisc_feature 1..140 /note= “TN-78 (normal)” 5 AGTGGAATTC CAACTTGAAGGATTGCATCC TGCTGGGGCT GAACATGCCT GCCAAAGAYG 60 TGTCCGACCT ACGTTCCTGGCCCCCTCGTT CAGAGACTGC CCTTCTCACG GGCTCTATGC 120 CTGCACTGGG AAGGAAACAA140 140 base pairs nucleic acid double linear DNA (genomic) Homo sapiensmisc_feature 1..140 /note= “TN-50 (tumor)” 6 AGTGGAATTC CAACTTGAAGGATTGCATCC TGCTGGGGCT GAACATGCCT GCCAAAGATG 60 TGTCTGACCT ACGTTCCTGGCCCCCTCGTT CAGAGACTGC CCTTCTCACG GGCTCTATGC 120 CTGCACTGGG AAGGAAACAA140 140 base pairs nucleic acid double linear DNA (genomic) Homo sapiensmisc_feature 1..140 /note= “TN-50 (normal)” 7 AGTGGAATTC CAACTTGAAGGATTGCATCC TGCTGGGGCT GAACATGCCT GCCAAAGAYG 60 TGTCCGACCT ACGTTCCTGGCCCCCTCGTT CAGAGACTGC CCTTCTCACG GGCTCTATGC 120 CTGCACTGGG AAGGAAACAA140 137 base pairs nucleic acid double linear DNA (genomic) Homo sapiens8 AGTGGAATTC CAACTTGAAG TATTGAATCC TTCTGGGGCT AAACATGCCT GCCAAAGATG 60TGTACATCCT GTGTTCCTGG CTTCCTTGTT CAGAGACTGC TCTTCTCCAG GGCTCTGTGC 120CTGTGCTTTG AAAACAG 137 142 base pairs nucleic acid double linear DNA(genomic) Homo sapiens misc_feature 1..142 /note= “TN-1 (tumor)” 9GGAATAATTC CAAGCTTGAA TGTCCAATCC TTCTGGGGTT TCTAAAGATC CTGCCAAAGA 60TGTGTACATC CTGTGTTCCT GGCTTCCTTG TTCGAGAACG ACTCTTCTCC ACGGCTCTGT 120GCCTGTGCTT TGAAGGAAAC AA 142 140 base pairs nucleic acid double linearDNA (genomic) Homo sapiens misc_feature 1..140 /note= “TN-1 (normal)” 10AGTGGAATTC CAACTTGAAG GATTGCATCC TGCTGGGGCT GAACATGCCT GCCAAAGAYG 60TGTCCGACCT ACGTTCCTGG CCCCCTCGTT CAGAGACTGC CCTTCTCACG GGCTCTATGC 120CTGCACTGGG AAGGAAACAA 140 135 base pairs nucleic acid double linear DNA(genomic) Homo sapiens misc_feature 1..135 /note= “TN-3 (tumor)” 11AATGGAATTC CAACTTGAAG TATTGAATCC TTCTGGCTAA ACATGCCTGC CAAAGATGTG 60TACATCCTGT GTTCCTGGCT TCCTTGTTCA GAGACTGCTC TTCTCCAGGG CTCTGTGCCT 120GTGCAAAGAA AATAG 135 140 base pairs nucleic acid double linear DNA(genomic) Homo sapiens misc_feature 1..140 /note= “TN-3 (normal)” 12AGTGGAATTC CAACTTGAAG GATTGCATCC TGCTGGGGCT GAACATGCCT GCCAAAGAYG 60TGTCCGACCT ACGTTCCTGG CCCCCTCGTT CAGAGACTGC CCTTCTCACG GGCTCTATGC 120CTGCACTGGG AAGGAAACAA 140 137 base pairs nucleic acid double linear DNA(genomic) Homo sapiens misc_feature 1..137 /note= “TN-31 (tumor)” 13AATGGAATTC CAACTTGAAG TATTGAATCC TTCTGGGGCT AAACATGCCT GCCAAAGATG 60TGTACATCCT GTGTTCCTGG CTTCCTTGTT CAGAGACTGC TCTTCTCCAG GGCTCTGTGC 120CTGTGCTTTG AAAATAG 137 140 base pairs nucleic acid double linear DNA(genomic) Homo sapiens misc_feature 1..140 /note= “TN-31 (normal)” 14AGTGGAATTC CAACTTGAAG GATTGCATCC TGCTGGGGCT GAACATGCCT GCCAAAGAYG 60TGTCCGACCT ACGTTCCTGG CCCCCTCGTT CAGAGACTGC CCTTCTCACG GGCTCTATGC 120CTGCACTGGG AAGGAAACAA 140 140 base pairs nucleic acid double linear DNA(genomic) Homo sapiens misc_feature 1..140 /note= “TN-94 (tumor)” 15AATGGAATTC CTTCTTGAAG TATTGAATCC TTCTGGGGCT AAACATGCCT GCCAAAGATG 60TGTACATCCT GTGTTCCTGG CTTCCTTGTT CAGAGACTGC TCTTGTCCAG GGCTCTGTGC 120CTGTGGTTTG AAGGAAACAA 140 140 base pairs nucleic acid double linear DNA(genomic) Homo sapiens misc_feature 1..140 /note= “TN-94 (normal)” 16AGTGGAATTC CAACTTGAAG GATTGCATCC TGCTGGGGCT GAACATGCCT GCCAAAGAYG 60TGTCCGACCT ACGTTCCTGG CCCCCTCGTT CAGAGACTGC CCTTCTCACG GGCTCTATGC 120CTGCACTGGG AAGGAAACAA 140 852 base pairs nucleic acid double linear DNA(genomic) Homo sapiens 17 CCCAGAAATC ACTGTGAAAT TTCATGATTG GCTTAAAGTGAAGGAAATAA AGGTAAAATC 60 ACTTCAGATC TCTAATTAGT CTATCAAATG AAACTCTTTCATTCTTCTCA CATCCATCTA 120 CTTTTTTATC CACCTCCCTA CCAAAAATTG CCAAGTGCCTATGCAAACCA GCTTTAGGTC 180 CCAATTCGGG GCCTGCTGGA GTTCCGGCCT GGGCACCAGCATTTGGCAGC ACGCAGGCGG 240 GGCAGTATGT GATGGACTGG GGAGCACAGG TGTCTGCCTAGATCCACGTG TGGCCTCCGT 300 CCTGTCACTG ATGGAAGGTT TGCGGATGAG GGCATGTGCGGCTGAACTGA GAAGGCAGGC 360 CTCCGTCTTC CCAGCGGTTC CTGTGCAGAT GCTGCTGAAGAGAGGTGCCG GGGAGGGGCA 420 GAGAGGAAGT GGTCTGTCTG TTACCATAAG TCTGATTCTCTTTAACTGTG TGACCAGCGG 480 AAACAGGTGT GTGTGAACTG GGCACAGATT GAAGAATCTGCCCCTGTTGA GGTGGGTGGG 540 CCTGACTGTT GCCCCCCAGG GTCCTAAAAC TTGGATGGACTTGTATAGTG AGAGAGGAGG 600 CCTGGACCGA GATGTGAGTC CTGTTGAAGA CTTCCTCTCTACCCCCCACC TTGGTCCCTC 660 TCAGATACCC AGTGGAATTC CAACTTGAAG GATTGCATCCTGCTGGGGCT GAACATGCCT 720 GCCAAAGACG TGTCCGACCT ACGTTCCTGG CCCCCTCGTTCAGAGACTGC CCTTCTCACG 780 GGCTCTATGC CTGCACTGGG AAGGAAACAA ATGTGTATAAACTGCTGTCA ATAAATGACA 840 CCCAGACCTT CC 852 21 base pairs nucleic acidsingle linear other nucleic acid /desc = “DNA primer” 18 TGAGTCCTGTTGAAGACTTC C 21 20 base pairs nucleic acid single linear other nucleicacid /desc = “DNA primer” YES 19 GGAAGGTCTG GGTGTCATTT 20 16 base pairsnucleic acid single linear other nucleic acid /desc = “DNA primer” 20CCCAGAAATC ACTGTG 16 16 base pairs nucleic acid single linear othernucleic acid /desc = “DNA primer” 21 GGATGGACTT GTATAG 16 1328 basepairs nucleic acid double linear DNA (genomic) 5′clip 1..477 22AGGACTGGTG GGCAATGTGC TCTGCTTCCC CCCGCTTCCC CCGCTAGCCA TCAGGAGGAA 60GTAAACTCCC CGAGTTCCTT CAGGAGCCTG GGAAGGTGGC TTTCTGGTGA AGGGCCTTTG 120GTTGTAGCCT GACATGCGGT GCCCTGAGGT TTGATCTTTG TCTCCACCTC CATTCTTTTA 180GGCTGAGCAA CAGAAAAAGG CGGCCATCAT CTCTGCTGAG GGCGACTCCA AGGCAGCTGA 240GCTGATTGCC AACTCACTGG CCACTGCAGG GGATGGCCTG ATCGAGCTGC GCAAGCTGGA 300AGCTGCAGAG GACATCGCGT ACCAGCTCTC ACGCTCTCGG AACATCACCT ACCTGCCAGC 360GGGGCAGTCC GTGCTCCTCC AGCTGCCCCA GTGAGGGCCC ACCCTGCCTG CACCTCCGCG 420GGCTGACTGG GCCACAGCCC CGATGATTCT TAACACAGCC TTCCTTCTGC TCCCACCCCA 480GAAATCACTG TGAAATTTCA TGATTGGCTT AAAGTGAAGG AAATAAAGGT AAAATCACTT 540CAGATCTCTA ATTAGTCTAT CAAATGAAAC TCTTTCATTC TTCTCACATC CATCTACTTT 600TTTATCCACC TCCCTACCAA AAATTGCCAA GTGCCTATGC AAACCAGCTT TAGGTCCCAA 660TTCGGGGCCT GCTGGAGTTC CGGCCTGGGC ACCAGCATTT GGCAGCACGC AGGCGGGGCA 720GTATGTGATG GACTGGGGAG CACAGGTGTC TGCCTAGATC CACGTGTGGC CTCCGTCCTG 780TCACTGATGG AAGGTTTGCG GATGAGGGCA TGTGCGGCTG AACTGAGAAG GCAGGCCTCC 840GTCTTCCCAG CGGTTCCTGT GCAGATGCTG CTGAAGAGAG GTGCCGGGGA GGGGCAGAGA 900GGAAGTGGTC TGTCTGTTAC CATAAGTCTG ATTCTCTTTA ACTGTGTGAC CAGCGGAAAC 960AGGTGTGTGT GAACTGGGCA CAGATTGAAG AATCTGCCCC TGTTGAGGTG GGTGGGCCTG 1020ACTGTTGCCC CCCAGGGTCC TAAAACTTGG ATGGACTTGT ATAGTGAGAG AGGAGGCCTG 1080GACCGAGATG TGAGTCCTGT TGAAGACTTC CTCTCTACCC CCCACCTTGG TCCCTCTCAG 1140ATACCCAGTG GAATTCCAAC TTGAAGGATT GCATCCTGCT GGGGCTGAAC ATGCCTGCCA 1200AAGACGTGTC CGACCTACGT TCCTGGCCCC CTCGTTCAGA GACTGCCCTT CTCACGGGCT 1260CTATGCCTGC ACTGGGAAGG AAACAAATGT GTATAAACTG CTGTCAATAA ATGACACCCA 1320GACCTTCC 1328 20 base pairs nucleic acid single linear other nucleicacid /desc = “DNA primer” 23 AAGGTGGCTT TCTGGTGAAG 20 19 base pairsnucleic acid single linear other nucleic acid /desc = “DNA primer” 24GGCCTCCGTC CTGTCACTG 19 20 base pairs nucleic acid single linear othernucleic acid /desc = “DNA primer” 25 CTTTGGCAGG CATGTTCAGC 20

We claim:
 1. A method for determining risk of a hereditary breastcancer, comprising the steps of: a. determining the base identity of aportion of genomic DNA from a patient cell sample, said genomic DNAcomprising a prohibitin gene comprising a 3′ untranslated region, saidportion corresponding to position 729 as defined in SEQ ID NO:17 of saidprohibitin gene in said 3′ untranslated region; and b. correlating saidbase identity with a risk for hereditary breast cancer.
 2. The method ofclaim 1, wherein the base identity of position 729 is determined bysequencing a portion of said portion of 3′ untranslated region of saidprohibitin gene containing said position
 729. 3. The method of claim 1,wherein base identity of said position 729 is determined by detection ofsingle base matches or mismatches between said portion of 3′untranslated region and C-allele prohibitin.
 4. The method of claim 1,wherein the base identity of position 729 is determined by digestingsaid portion of 3′ untranslated region of said prohibitin gene with arestriction endonuclease appropriate to determine the base identity ofsaid position
 729. 5. The method of claim 4, wherein said restrictionendonuclease is AflIII, and whereby it is determined that a cleavagesite affected by AflIII is present when position 729 is cytosine.
 6. Themethod of claim 5, further comprising the steps of: a. separating saiddigested portion of 3′ untranslated region DNA strands; b. fixing saidseparated digested 3′ untranslated region DNA strands onto a membrane;c. hybridizing said separated digested 3′ untranslated region DNAstrands with at least one labeled nucleic acid probe, wherein saidlabeled nucleic acid probe can complementarily bind to said fixedseparated digested 3′ untranslated region DNA strands and can identifywhether cleavage at said position 729 occurred; and d. detecting if saidlabeled nucleic acid probe has bound to said fixed separated digested 3′untranslated region DNA strands, wherein said patient is at risk forhereditary breast cancer if said labeled nucleic acid probe bound tosaid fixed separated digested 3′ untranslated region DNA strandsindicates cleavage at said position 729 did not occur.
 7. The method ofclaim 1, wherein said base identity is determined by examining an RNAfraction from said patient cell sample, whereby the identity of saidgenomic DNA at said position 729 can be determined.
 8. The method ofclaim 1, wherein a lifetime risk for developing breast cancer isassessed to be greater than that of the unaffected relevant populationwhen the base identity at said position 729 is homozygous for thymine.9. The method of claim 1, wherein a lifetime risk for developing breastcancer is assessed to be greater than that of the unaffected relevantpopulation but less than that of an individual who is homozygous forthymine when the base identity at said position 729 is heterozygouscytosine/thymine.
 10. The method of claim 1, wherein a lifetime risk fordeveloping breast cancer is assessed to be less than or equal to theunaffected relevant population when the base identity at said position729 is homozygous cytosine.
 11. A method for determining the risk forhereditary breast cancer in a human patient, comprising the steps of: a.isolating a portion of double-stranded genomic DNA from a patient cellsample, said genomic DNA comprising a prohibitin gene comprising a 3′untranslated region; b. separating said double-stranded genomic DNA intoa first single-stranded genomic DNA and a second single-stranded genomicDNA in a first reaction zone; c. providing a sense primer to saidreaction zone, said reaction zone having conditions favorable forhybridization between said first single-stranded genomic DNA and saidsense primer; d. simultaneously providing an antisense primer to saidreaction zone, said reaction zone having conditions favorable forhybridization between said second single-stranded genomic DNA and saidantisense primer; e. making multiple copies of said portion ofdouble-stranded genomic DNA by polymerase chain reaction methodology toform synthesized double-stranded DNA; f. determining the base identityof position 729 as defined by SEQ ID NO:17 for said 3′ untranslatedregion DNA strands; and g. correlating said base identity with a riskfor hereditary breast cancer, wherein said patient is at lowest riskwith homozygous C/C, intermediate risk with heterozygous C/T, andgreatest risk with homozygous T/T at said position
 729. 12. The methodof claim 11, wherein said sense primer comprises SEQ ID NO:18.
 13. Themethod of claim 11, wherein said sense primer-comprises SEQ ID NO:20.14. The method of claim 11, wherein said sense primer comprises SEQ IDNO:21.
 15. The method of claim 11, wherein said sense primer comprisesSEQ ID NO:23.
 16. The method of claim 11, wherein said antisense primercomprises SEQ ID NO:19.
 17. The method of claim 12, wherein saidantisense primer comprises SEQ ID NO:19.
 18. The method of claim 13,wherein said antisense primer comprises SEQ ID NO:19.
 19. The method ofclaim 14, wherein said antisense primer comprises SEQ ID NO:19.
 20. Themethod of claim 15, wherein said antisense primer comprises SEQ IDNO:19.
 21. The method of claim 18, further comprising, prior to step f,purifying to form an 852 bp fragment and performing secondary polymerasechain reaction using sense primer comprising SEQ ID NO:18 and antisenseprimer comprising SEQ ID NO:19 to form synthesized double-stranded DNA.22. The method of claim 11, wherein base identity of said position 729is determined by sequencing.
 23. The method of claim 11, wherein baseidentity of said position 729 is determined by detection of single basematches or mismatches between said synthesized double-strand DNA andC-allele prohibitin.
 24. The method of claim 11, wherein base identityof said position 729 is determined by restriction fragment lengthpolymorphism.
 25. The method of claim 11 further comprising digestingsaid synthesized double-stranded DNA with restriction endonucleaseAflIII which cleaves said untranslated region at said base 729 when saidbase is cytosine.
 26. The method of claim 21 further comprisingdigesting said synthesized double-stranded DNA with restrictionendonuclease AflIII which cleaves said untranslated region at said base729 when said base is cytosine.
 27. The method of claim 25, furthercomprising the steps of: h. separating said digested synthesizeddouble-stranded DNA strands; i. fixing said separated digestedsynthesized double-stranded DNA strands onto a membrane; j. hybridizingsaid separated digested synthesized double-stranded DNA strands with atleast one labeled nucleic acid probe, wherein said labeled nucleic acidprobe can complementarily bind to said fixed separated digestedsynthesized double-stranded DNA strands and can identify whethercleavage at said position 729 occurred; and k. detecting if said labelednucleic acid probe has bound to said fixed separated digestedsynthesized double-stranded DNA strands, wherein said patient is at riskfor hereditary breast cancer if said labeled nucleic acid probe bound tosaid fixed separated digested synthesized double-stranded DNA strandsindicates cleavage at said position 729 did not occur.
 28. The method ofclaim 25, further comprising the steps of: h. separating said digestedsynthesized double-stranded DNA strands; and i. visualizing saiddigested synthesized double-stranded DNA fragment pattern by ethidiumbromide staining and ultraviolet photography.
 29. The method of claim26, further comprising the steps of: h. separating said digestedsynthesized double-stranded DNA strands; i. fixing said separateddigested synthesized double-stranded DNA strands onto a membrane; j.hybridizing said separated digested synthesized double-stranded DNAstrands with at least one labeled nucleic acid probe, wherein saidlabeled nucleic acid probe can complementarily bind to said fixedseparated digested synthesized double-stranded DNA strands and canidentify whether cleavage at said position 729 occurred; and k.detecting if said labeled nucleic acid probe has bound to said fixedseparated digested synthesized double-stranded DNA strands, wherein saidpatient is at risk for hereditary breast cancer if said labeled nucleicacid probe bound to said fixed separated digested synthesizeddouble-stranded DNA strands indicates cleavage at said position 729 didnot occur.
 30. The method of claim 26, further comprising the steps of:h. separating said digested synthesized double-stranded DNA strands; andi. visualizing said digested synthesized double-stranded DNA fragmentpattern by ethidium bromide staining and ultraviolet photography. 31.The method of claim 11, wherein said portion of genomic DNA is SEQ IDNO:22.
 32. The method of claim 31, wherein said sense primer SEQ IDNO:23 is used to amplify SEQ ID NO:22.
 33. The method of claim 31,wherein said antisense primer SEQ ID NO:19 is used to amplify SEQ IDNO:22.
 34. The method of claim 32, wherein said antisense primer SEQ IDNO:19 is used to amplify SEQ ID NO:22.
 35. The method of claim 34,wherein base identity of said position 729 is determined by sequencing.36. The method of claim 34, wherein base identity of said position 729is determined by detection of single base mismatches between saidsynthesized double-strand DNA and C-allele prohibitin.
 37. The method ofclaim 34, wherein base identity of said position 729 is determined byrestriction fragment length polymorphism.
 38. The method of claim 34further comprising digesting said synthesized double-stranded DNA withrestriction endonuclease AflIII which cleaves said untranslated regionat said base 729 when said base is cytosine.
 39. The method of claim 38,further comprising the steps of: h. separating said digested synthesizeddouble-stranded DNA strands; i. fixing said separated digestedsynthesized double-stranded DNA strands onto a membrane; j. hybridizingsaid separated digested synthesized double-stranded DNA strands with atleast one labeled nucleic acid probe, wherein said labeled nucleic acidprobe can complementarily bind to said fixed separated digestedsynthesized double-stranded DNA strands and can identify whethercleavage at said position 729 occurred; and k. detecting if said labelednucleic acid probe has bound to said fixed separated digestedsynthesized double-stranded DNA strands, wherein said patient is at riskfor hereditary breast cancer if said labeled nucleic acid probe bound tosaid fixed separated digested synthesized double-stranded DNA strandsindicates cleavage at said position 729 did not occur.
 40. The method ofclaim 38, further comprising the steps of: h. separating said digestedsynthesized double-stranded DNA strands; and i. visualizing saiddigested synthesized double-stranded DNA fragment pattern by ethidiumbromide staining and ultraviolet photography.
 41. A method fordetermining the risk for hereditary breast cancer in a human patientcomprising the steps of: a. determining the sequence of RNA isolatedfrom said patient in a region which is a transcription of a portion ofgenomic DNA, said genomic DNA comprising a prohibitin gene comprising anuntranslated region, said portion corresponding to position 729 asdefined in SEQ ID NO:17 of said prohibitin gene in said untranslatedregion; and b. correlating said base identity with a risk for hereditarybreast cancer.